Rotaviruses (RV) are the leading cause of severe gastroenteritis in infants and children worldwide. The RV genome consists of 11 segments of dsRNA which encode 6 structural (VP) and 6 non-structural (NSP) viral proteins. The diversity and complexity of RV antigens (outer capsid VP4, VP7, and NSP4 sero/genotypes) have hindered identification of the determinants of protective immunity. Both the complexity of RV and the need to induce intestinal immunity in infants have impeded development of effective RV vaccines. Because the exact nature and extent of cross-protection to RV serotypes is not clearly characterized, there is little consensus among researchers on the necessity for pursuing monovalent versus multivalent RV vaccines. The goal of our proposed studies is to improve our understanding of the immunological mechanisms of heterotypic compared to homotypic protective immunity and to identify the correlates of immunity for heterotypic protection. We will extend our studies of the neonatal gnotobiotic pig model of homotypic HRV infection and disease to determine the extent and mechanisms of cross-protective immunity between two distinct VP7(G) types, G1 (Wa, P1AG1) and G3 (M, P1AG3), the two most prevalent human RV (HRV) G serotypes identified in many epidemiologic surveys. A variety of virus-like particles (VLPs) which are antigenically similar to native double (2/6VLP) or triple (2/4/6/7VLP) layered RV particles but non-infectious will allow us to dissect the contributions of VP4, VP6, or NSP4 from those of VP7 in heterotypic (G type) protective immunity. Our specific aims are as follows. 1) To investigate the role of replicating versus non-replicating vaccines and the effect of vaccine routes (oral versus intranasal, IN) to prime neonatal mucosal immune responses and to induce protection. We will examine whether IN or oral 2/4/6/7VLPs can substitute for oral attenuated HRV in priming for protective immunity with 2/6VLP boosters and homotypic virulent HRV challenge. We will explore the mechanisms of VLP booster responses by in vivo and in vitro boosting of primed lymphocytes with VLPs. To test the effect of route, adjuvant, and preexisting immunity on vaccine uptake, we will investigate the uptake of green-fluorescent fusion protein-2/6VLP given orally or IN with or without immune stimulating complexes adjuvant (ISCOM, used for all VLPs) in naive versus 2/6VLP primed pigs. 2) To investigate the role of VP6 in heterotypic protection, we will compare immune responses and protection rates between pigs primed with G1 or G3 VLP or attenuated HRV vaccines and boosted with 2/6VLPs or 2/4/6/7VLPs and then challenged with virulent G3 or G1 HRV. 3) To determine the role of VP7 in heterotypic protection, we will add VP7 to the 2/6VLP booster vaccines (in the form of 2/6/7VLPs) to examine how repeated exposure to VP7 (without VP4) contributes to the immune responses to heterotypic HRV and protection. 4) To examine the role of VP4 in heterotypic immunity, we will compare VLP vaccines with or without VP4. Because Wa and M HRV share the same VP4 type, the contribution of VP4 in homotypic or heterotypic (G type) protection can be dissected from that of VP6 and VP7. 5) To examine the role of NSP4 in enhancing homotypic and heterotypic protection, we will evaluate if addition of NSP4 (in NSP4-VLPs) as booster vaccines enhances the immune responses and increases the protection rates to homotypic RV challenge or broadens the protection rates to heterotypic RV challenge. Immune responses (HRV-specific, RV-protein-specific, and memory T and B cells and antibodies, serotype-specific virus neutralizing antibodies, and cytokine profiles) will be quantitated in intestinal, tonsillar, and systemic lymphoid tissues using ELISPOT, LPA, FACS, neutralization tests, and ELISA. A better understanding of the mechanism of induction of heterotypic immunity to RV will facilitate the development of effective and safe vaccination strategies to confer consistent protection in the face of HRV infection by the major serotypes prevalent worldwide.